Chapter 13web.biosci.utexas.edu/psaxena/BIO226R/pdf/ch_13.pdf · 2006. 3. 11. · Title: Chapter 13...
Transcript of Chapter 13web.biosci.utexas.edu/psaxena/BIO226R/pdf/ch_13.pdf · 2006. 3. 11. · Title: Chapter 13...
![Page 1: Chapter 13web.biosci.utexas.edu/psaxena/BIO226R/pdf/ch_13.pdf · 2006. 3. 11. · Title: Chapter 13 Author: John Sherwood Created Date: 10/14/2005 4:25:20 PM](https://reader033.fdocuments.us/reader033/viewer/2022061001/60b022305e9c6c57fb34da3b/html5/thumbnails/1.jpg)
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Chapter 13
Microbial Recombination and Plasmids
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Eucaryotic recombination
• recombination– process in which one or
more nucleic acid molecules are rearranged or combined to produce a new nucleotide sequence
• in eucaryotes, usually occurs as the result of crossing-over during meiosis
Figure 13.1
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Bacterial Recombination: General Principles
• several types of recombination– general recombination
• can be reciprocal or nonreciprocal– site-specific recombination– replicative recombination
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Reciprocal general recombination• most common type of recombination• a reciprocal exchange between pair of
homologous chromosomes• results from DNA strand breakage and
reunion, leading to crossing-over
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5Figure 13.2
Reciprocal general recom-bination
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6Figure 13.2
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Nonreciprocal general recombination• incorporation of single
strand of DNA into chromosome, forming a stretch of heteroduplex DNA
• proposed to occur during bacterial transformation
Figure 13.3
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Site-specific recombination
• insertion of nonhomologous DNA into a chromosome
• often occurs during viral genome integration into host chromosome– enzymes responsible are specific for virus
and its host
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Replicative recombination
• accompanies replication of genetic material
• used by genetic elements that move about the genome
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Horizontal gene transfer
• transfer of genes from one mature, independent organism (donor) to another (recipient)
• exogenote– DNA that is transferred to recipient
• endogenote– genome of recipient
• merozyogote– recipient cell that is temporarily diploid as result of
transfer process
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11Figure 13.4
three mechanisms ofgene transfer
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Bacterial Plasmids• small, double-stranded, usually circular DNA
molecules• are replicons
– have their own origin of replication– can exist as single copies or as multiple copies
• curing– elimination of plasmid– can be spontaneous or induced by treatments that
inhibit plasmid replication but not host cell reproduction
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Bacterial plasmids…
• episomes– plasmids that can exist either with or without
integrating into chromosome• conjugative plasmids
– have genes for pili– can transfer copies of themselves to other
bacteria during conjugation
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Fertility Factors
• conjugative plasmids
• e.g., F factor of E. coli
• many are also episomes
Figure 13.5
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16Figure 13.7
mediated byinsertion sequences(IS)
F plasmid integration
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Resistance Factors
• R factors (plasmids)• have genes for resistance to antibiotics• some are conjugative• usually do not integrate into chromosome
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Col plasmids
• encode colicin – kills E. coli– a type of bacteriocin
• protein that destroys other bacteria, usually closely related species
• some are conjugative• some carry resistance genes
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Other Types of Plasmids• virulence plasmids
– carry virulence genes• e.g., genes that confer resistance to host
defense mechanisms• e.g., genes that encode toxins
• metabolic plasmids– carry genes for metabolic processes
• e.g., genes encoding degradative enzymes for pesticides
• e.g., genes for nitrogen fixation
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Transposable Elements
• transposition– the movement of pieces of DNA around the
genome• transposable elements (transposons)
– segments of DNA that carry genes for transposition
• widespread in bacteria, eucaryotes and archaea
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Types of transposable elements• insertion sequences (IS elements)
– contain only genes encoding enzymes required for transposition
• e.g., transposase
• composite transposons– carry genes in addition to those needed for
transposition– conjugative transposons
• carry transfer genes in addition to transposition genes
![Page 22: Chapter 13web.biosci.utexas.edu/psaxena/BIO226R/pdf/ch_13.pdf · 2006. 3. 11. · Title: Chapter 13 Author: John Sherwood Created Date: 10/14/2005 4:25:20 PM](https://reader033.fdocuments.us/reader033/viewer/2022061001/60b022305e9c6c57fb34da3b/html5/thumbnails/22.jpg)
22Figure 13.8
IR = inverted repeats
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The transposition event
• usually transposon replicated, remaining in original site, while duplicate inserts at another site
• insertion generates direct repeats of flanking host DNA
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25Figure 13.10
Tn3 trans-position
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26Figure 13.10
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27Figure 13.9
Generation of direct repeats
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Effects of transposition
• mutation in coding region– e.g., deletion of genetic material
• arrest of translation or transcription• activation of genes• generation of new plasmids
– e.g., resistance plasmids
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29Figure 13.11
R1 plasmid
RTF = resistancetransfer factor
a conjugativeplasmid
sources ofresistance genesare transposons
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Bacterial Conjugation
• transfer of DNA by direct cell to cell contact
• discovered 1946 by Lederberg and Tatum
Figure 13.12
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31Figure 13.13
The U-tube experiment
demonstrated thatdirect cell to cellcontact wasnecessary
after incubation,bacteria plated onminimal media
no prototrophs
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F+ x F– Mating• F+ = donor
– contains F factor• F– = recipient
– does not contain F factor• F factor replicated by rolling-circle mechanism
and duplicate is transferred• recipients usually become F+
• donor remains F+
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Figure 13.14a
F+ x F– mating
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Hfr Conjugation
• Hfr strain– donor having F factor integrated into its
chromosome• both plasmid genes and chromosomal
genes are transferred
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Figure 13.14b
Hfr x F– mating
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F′ Conjugation
• F′ plasmid– formed by
incorrect excision from chromosome
– contains ≥ 1 genes from chromosome
• F′ cell can transfer F′ plasmid to recipient
Figure 13.15a
integrated F factorchromosomal gene
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Figure 13.15b
F′ x F– mating
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DNA Transformation
• uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form
• competent cell– capable of taking up DNA
• may be important route of genetic exchange in nature
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39Figure 13.16a
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40Figure 13.16b
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DNA bindingprotein
nuclease – nicks and degrades onestrand
competence-specificprotein
Streptococcus pneumoniae
Figure 13.17
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Artificial transformation
• transformation done in laboratory with species that are not normally competent (e.g., E. coli)
• variety of techniques used to make cells temporarily competent– e.g., calcium chloride treatment
• makes cells more permeable to DNA
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Transduction
• transfer of bacterial genes by viruses• virulent bacteriophages
– reproduce using lytic life cycle• temperate bacteriophages
– reproduce using lysogenic life cycle
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Phage lambda life cycles
Figure 13.18 prophage=integrated form of viral genome
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Generalized Transduction
• any part of bacterial genome can be transferred
• occurs during lytic cycle• during viral assembly, fragments of host
DNA mistakenly packaged into phage head– generalized transducing particle
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Figure 13.19
Generalized transduction
abortive transductants- bacteria with nonintegrated transduced DNA
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Specialized Transduction
• also called restricted transduction• carried out only by temperate phages
that have established lysogeny• only specific portion of bacterial genome
is transferred• occurs when prophage is incorrectly
excised
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48Figure 13.20
Specialized transduction
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Figure 13.20
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Phage lambda
Figure 13.21
low-frequencytransducinglysate
insert Figure 13.21
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Figure 13.21 high-frequency transduction lysate
insert Figure 13.21
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Mapping the Genome
• locating genes on an organism’s chromosomes
• mapping bacterial genes accomplished using all three modes of gene transfer
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Hfr mapping
• used to map relative location of bacterial genes
• based on observation that chromosome transfer occurs at constant rate
• interrupted mating experiment– Hfr x F- mating interrupted at various intervals– order and timing of gene transfer determined
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54Figure 13.22a
Interrupted mating
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55Figure 13.22b
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E. coli genetic map
• gene locations expressed in minutes, reflecting time transferred
• made using numerous Hfr strains
Figure 13.23
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57
Transformation mapping
• used to establish gene linkage• expressed as frequency of
cotransformation• if two genes close together, greater
likelihood will be transferred on single DNA fragment
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58
Generalized transduction mapping• used to establish gene linkage• expressed as frequency of cotransduction• if two genes close together, greater
likelihood will be carried on single DNA fragment in transducing particle
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59
Specialized transduction mapping• provides distance of genes from viral
genome integration sites• viral genome integration sites must first be
mapped by conjugation mapping techniques
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Recombination and Genome Mapping in Viruses
• viral genomes can also undergo recombination events
• viral genomes can be mapped by determining recombination frequencies
• physical maps of viral genomes can also be constructed using other techniques
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Recombination mapping• recombination
frequency determined when cells infected simultaneously with two different viruses
Figure 13.24
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Physical maps
• heteroduplex maps– genomes of two different viruses denatured,
mixed and allowed to anneal• regions that are not identical, do not reanneal
– allows for localization of mutant alleles
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Physical maps…
• restriction endonuclease mapping– compare DNA fragments from two different
viral strains in terms of electrophoretic mobility
• sequence mapping– determine nucleotide sequence of viral
genome– identify coding regions, mutations, etc.